Description
Pyruvate Kinase Activity Assay Kit (BA0145) (BA0145)
The Pyruvate Kinase Activity Assay Kit (SKU: BA0145) provides a simple, direct and automation-ready procedure for measuring pyruvate kinase (PK) activity. Pyruvate kinase is an enzyme involved in glycolysis that catalyses the transfer of a phosphate group from phosphoenolpyruvate to ADP, yielding pyruvate and ATP, and it also participates in gluconeogenesis. In this assay phosphoenolpyruvate and ADP are catalysed by pyruvate kinase to generate pyruvate and ATP, and the pyruvate generated is measured through a coupled colour or fluorescence reaction. The colour intensity at 570 nm, or the fluorescence intensity at 530/590 nm, is directly proportional to the pyruvate kinase activity in the sample. The assay is sensitive and accurate and is readily automated for high-throughput use.
| Product Name: | Pyruvate Kinase Activity Assay Kit (BA0145) |
| SKU: | BA0145 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 0.1 - 50 U/L; fluorometric 0.01 - 2 U/L |
| Sample Type: | Plasma, serum and tissue samples |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all kit components at -20 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A coupled enzymatic assay for the quantitative colorimetric or fluorometric determination of pyruvate kinase activity. Pyruvate kinase converts phosphoenolpyruvate and ADP into pyruvate and ATP; the pyruvate generated is measured through a colour reaction at 570 nm or a fluorescence reaction at 530/590 nm. The method is direct and suitable for automation.
- Sensitive and accurate
- Linear detection range in 96-well plate: colorimetric 0.1 - 50 U/L, fluorometric 0.01 - 2 U/L (30 min at 25 C)
- Direct, automation-ready procedure
- Choice of colorimetric or fluorometric detection
- Measurement of pyruvate kinase activity in plasma, serum and tissue samples
- Studies of the effects of drugs on pyruvate kinase activity
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Equilibrate the Developer to the desired assay temperature. |
| 2 | Prepare samples: homogenise tissue or cells (2x10^6) in 100 uL PBS and centrifuge at 14,000 rpm for 5 min; use the clear supernatant. Dilute serum at least 4-fold in water. |
| 3 | Prepare standards: dilute the Pyruvate Standard to 1000 uM (20 uL of 25 mM in 480 uL water), then dilute to 1000, 600, 300 and 0 uM. Transfer 10 uL of each standard and 10 uL of each sample into wells of a clear flat-bottom 96-well plate (or a black plate for the fluorometric assay, using a 1:20 dilution of the standards). |
| 4 | Prepare Working Reagent per well by mixing 95 uL Developer, 1 uL Cosubstrate and 1 uL Dye Reagent. Add 90 uL to each standard and sample well, mix and incubate protected from light. |
| 5 | Read optical density at 570 nm at 5 min and again at 35 min (colorimetric), or read fluorescence at 530/590 nm at 5 and 35 min (fluorometric). |
Using the 35 min readings, subtract the blank and plot the change in optical density or fluorescence against pyruvate concentration to determine the slope. For each sample subtract the 5 min reading from the 35 min reading, then calculate [PK] = ((dR_SAMPLE - dR_BLANK) / (Slope x t)) x n (U/L), where t is the reaction time (30 min) and n is the dilution factor. One unit of PK generates 1 umole of pyruvate and 1 umole of ATP per minute at 25 C and pH 7.5.
| Component | Quantity | Storage |
| Developer | 12 mL | -20 C |
| Cosubstrate | 120 uL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Pyruvate Standard (25 mM) | 400 uL | -20 C |
